Figure 1. Nucleotide excision repair in eukaryotic cells. (a) The nucleotide excision repair machinery (repairosome) is stably bound to DNA at a site of base damage involving two adjacent nucleotides (filled triangle). The base damage itself is associated with some distortion of the duplex structure of DNA. The yeast Rad3 and Ssl2 DNA helicases in the TFIIH subcomplex facilitate localized unwinding of the DNA flanking the site of base damage, generating a denaturation ‘bubble’ about 30 nucleotides in length (b). This process is associated with a specific conformational change in the structure of the repairosome–DNA complex. The junctions between duplex and single-stranded DNA at each margin of the bubble are now recognized by the active sites of the Rad1/Rad10 and Rad2 endonucleases, which incise the strand carrying the damaged base (c). In this manner an oligonucleotide fragment is generated, the displacement of which yields a (potential) gap in the DNA duplex that can then be filled in by the action of a DNA polymerase using the opposite intact strand as a template. Following repair synthesis the covalent integrity of the repaired strand is restored by the action of a DNA ligase.

Figure 2. Autoradiogram showing repair synthesis of DNA associated with nucleotide excision repair in human cells in culture. (a) Repair synthesis in non-S phase normal cells. Cells that are in the S phase of the cell cycle are intensely labelled. (b) Defective repair synthesis in cells from an individual with the hereditary disease xeroderma pigmentosum (XP).

Figure 3. Assay used for measuring nucleotide excision repair in cell-free extracts. The assay monitors repair synthesis of plasmid DNA previously treated with acetylaminofluorene (AAF) and incubated in the presence of a radiolabelled deoxynucleoside triphosphate (dCTP). Radioactivity incorporated into the damaged plasmid during NER is observed by autoradiography following linearization and electrophoresis of the plasmid. A second (larger) plasmid not treated with AAF and which migrates more slowly in the gel during electrophoresis, is included in the assay to monitor background levels of nonspecific DNA synthesis. (Red triangles denote sites of base damage in plasmid DNA. The red squares are sites of incorporation of radiolabelled dCMP during NER. These sites are revealed as a discrete signal (red band) following autoradiography of gels.)

Figure 4. Extracts of a wild-type yeast strain are proficient in repair as evidenced by the autoradiographic signal in the AAF-treated plasmid DNA (+AAF) relative to the control plasmid (–AAF) (lanes 5 and 6, bottom panel). Extracts of either ssl2 mutants (lanes 3 and 4) or rad10 mutants (lanes 1 and 2, bottom panel) are defective in NER. The top panel shows the gel stained with ethidium bromide prior to autoradiography in order to demonstrate that approximately equal amounts of plasmid DNA were loaded into each of the lanes.